Heart failure remains the leading cause of death for people over the age of 65 in the US. Oxidative damage to mitochondrial proteins is thought to play an important role in decline of mitochondrial function and myocardial decay during the aging process, but the nature and extent of damage to mitochondrial proteins as a result of aging is largely unknown. The overall hypothesis to be evaluated is that damage to mitochondrial proteins as a result of adduction of lipid peroxidation products and glutathionylation contributes to loss of mitochondrial function in the aging heart. To test this hypothesis, a functional proteomics approach will be used based on identification and quantification of proteins by mass spectrometry and analysis of the data by multivariate methods. Specific Aims: (1) Selective detection of mitochondrial proteins that contain free, exposed cysteines by using a thiol-specific probe with the aim to identify thiol proteins that are potential targets of oxidative damage. (2) To characterize and quantify adduction of lipid peroxidation products to thiol proteins. (3) To identify and quantify glutathionylated proteins. (4) To determine the activity of mitochondrial enzymes that are highly modified by lipid peroxidation products or by glutathionylation during aging, with the aim to correlate the proteomic data with changes in enzyme activity. The levels of oxylipid adduction and glutathionylation of mitochondrial thiol proteins are anticipated to be low, and therefore specific isotope-coded affinity tags will be used for enrichment of these modified proteins. Protein quantification will be achieved with isotope-dilution mass spectrometry by comparing mitochondria from young and old rat hearts. Significance: These studies will provide unique insights relating how specific protein modification may impact mitochondrial enzyme function by using novel mass spectrometry-based functional proteomics approaches. The results will be the basis for our long-term goals to identify unique mitochondrial protein targets for oxidative modification that affect overall organelle and cardiac function in the elderly.